Severe acute respiratory distress syndrome (SARS) is a new respiratory disorder in humans that is caused by the SARS coronavirus (SARS-CoV). The disease emerged at the beginning of 2003 in China and various other portions of South East Asia and has since then rapidly spread throughout the world. Although the disease had disappeared by June 2003 its re-emergence cannot be excluded. Therefore, much effort is currently being put into the development of therapeutic and prophylactic treatments for SARS-CoV.
Although SARS-CoV is phylogenetically distinct from all previously known human and animal coronaviruses, significant progress has been made in understanding the molecular and cell biology of SARS-CoV. Next to the complete sequence of the SARS-CoV genome (see Marra et al. (2003); Rota et al. (2003)), Li et al. (2003) have recently identified a zinc metallopeptidase, angiotensin-converting enzyme 2 (ACE2 protein), as a functional receptor for SARS-CoV. This and other knowledge regarding the molecular and cell biology of SARS-CoV have offered avenues for developing anti-viral as well as vaccine strategies.
The development of a vaccine protecting against SARS-CoV has mainly focused on two strategies, i.e. the use of inactivated whole SARS-CoV (Tang et al. (2004); Takasuka et al. (2004)) and the use of SARS-CoV proteins (Zhang et al. (2004); Yang et al. (2004); Kim et al. (2004)). Inactivated whole virus vaccines are usually prepared by producing large amounts of virus in cell tissue culture and then rendering the virus harmless without destroying its immunological properties. For optimal virus production in cell culture, it is pivotal that the respective virus is capable of infecting the cells and replicating in the cell. To date only a limited number of cells have been reported to be susceptible to SARS-CoV infection and to support SARS-CoV replication in culture (see Mossel et al. (2005)). The most frequently used cells in that respect are kidney cells derived from African Green Monkeys such as Vero or Vero E6 cells. A disadvantage associated with these cells is inter alia that they require the presence of serum and/or the adherence to a solid support for growth resulting in purification and safety issues as well as a laborious system for large-scale production. Furthermore, the cells are not human.
Recently, it was shown that cells refractory to SARS-CoV infection could be rendered permissive for SARS-CoV replication by expressing a functional receptor, i.e. the human ACE2 receptor. In WO 2005/032487 it was shown that human 293T cells transfected with the ACE2 protein supported SARS-CoV replication and were suitable for the production of SARS-CoV. However, the yields obtained with these cells were low making production methods using them economically unattractive. Taken together, there is still a need in the art for a method of producing SARS-CoV in a host cell system that improves on the existing cell culture systems, specifically on the yields obtained.
The present invention addresses this need by providing primary human retina cells (HER cells) expressing the ACE2 protein. These cells give unexpectedly high SARS-CoV yields. They have as a further advantage that they are extensively documented and better behave in the process of upscaling, suspension growth and growth factor independence compared to the cells in the art. Especially the fact that the cells can be brought in suspension in a highly reproducible manner is something that makes them very suitable for large scale production. Moreover, the cells of the present invention can advantageously be used for the replication of various isolates of human SARS-CoV and are further not only suitable for the production of SARS-CoV, but also for production of other human coronaviruses that make use of the ACE2 protein as a functional receptor.